Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase

Abstract

The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH.

Figure 1.

Increased Biomass Does Not Require Constitutive AVP1 Overexpression. (A) Increased AVP1 protein amounts in UBQ:AVP1 lines. Microsomal membrane extracts of 4-week-old wild-type, 35S:AVP1, and UBQ:AVP1 plants were separated by SDS-PAGE and subsequently immunoblotted with anti-V-PPase antibody. Equal protein loading is indicated by anti-VHA-C detection. (B) Elevated K⁺-stimulated PPase activity in UBQ:AVP1 but not in 35S:AVP1 lines. Plants were grown for 4 weeks under LD conditions. Wild type activity was set to 100%. Graph shows result of one representative experiment of three biological replicates. Error bars indicate sd of n = 3 technical replicates. (C) and (D) Phenotypes (C) and rosette fresh weight (D) of 4-week-old 35S:AVP1 plants compared with UBQ:AVP1 lines grown under LD conditions. Error bars indicate se of n = 8 to 11 plants. FW, fresh weight. Bar = 3.5 cm.

Figure 2.

Overexpression of AVP1 Does Not Complement the Tonoplast V-ATPase Double Mutant vha-a2 vha-a3. (A) Arabidopsis wild-type and vha-a2 vha-a3 mutant plants expressing AVP1 under the UBQ promoter have higher AVP1 protein level. Microsomal membrane proteins of 3-week-old plants were extracted, separated by SDS-PAGE, and subsequently immunoblotted with anti-V-PPase antibody. Equal protein loading is indicated by TIP1;1 detection. A quantification of AVP1 protein levels is shown below and each bar corresponds with the band in the blot immediately above it. Error bars indicate se of n = 3 technical replicates. (B) UBQ:AVP1 cannot restore wild type growth in vha-a2 vha-a3. Plants were grown for 3 weeks under LD conditions at 22°C. Bar = 1.75 cm. (C) AVP1 overexpression lines show increased V-PPase activity. K⁺-stimulated PPase activity was determined using microsomal membranes extracted from 3-week-old plants. Wild type activity was set to 100%. Error bars represent se of n = 3 biological replicates. (D) Overexpression of AVP1 has no effect on cell sap pH. Plants for cell sap pH measurements were grown for 3 weeks under LD conditions at 22°C. Error bars show sd of n = 3 biological replicates. (E) Vacuolar pH in root epidermal cells is not changed upon AVP1 overexpression. Vacuolar pH was measured in roots of 6-d-old plants. Error bars represent sd of n = 3 biological replicates.

Figure 3.

T-DNA Insertion in GNOM Causes Phenotypic Defects in avp1-1. (A) Position of the two T-DNA Insertions on chromosome 1 in the GABI-Kat 005D04 line. RB, right border; LB, left border. Bars = 500 bp. (B) Comparison of the phenotypes of 5-d-old wild-type, fugu5-1, avp1-1, and gnom^R5 seedlings. Bar = 1 cm. (C) Allelism test between V-PPase and gnom mutants. V-PPase mutants (vhp1-1 and fugu5-1) were crossed with avp1-1/+ or with gnom/+ (emb30-1/+) mutants. Phenotypes of 6-d-old F1 seedlings (n = 80 to 190) were counted. Numbers given in the table are percentages (%).

Figure 4.

Increase of V-ATPase Activity after Cold Acclimation Depends on V-PPase. (A) Lack of V-PPase activity prevents full acidification upon cold acclimation. Plants for cell sap pH measurements were grown for 6 weeks under short-day conditions at 22°C. Error bars represent sd of n = 3 biological replicates. Asterisk indicates significant difference at P < 0.05 (Student’s t test). (B) V-PPase activity affects the induction of V-ATPase activity upon cold acclimation. KNO₃-inhibited ATPase activity was determined with microsomal membranes extracted from 6-week-old, short day-grown plants that were cold-acclimated for 4 d at 4°C. Error bars show sd of n = 3 biological replicates. Asterisk indicates significant difference at P < 0.05 (Student’s t test). (C) V-PPase activity of the wild type and UBQ:AVP1 increases after cold exposure. K⁺-stimulated PPase activity was determined from microsomal membranes extracted from 6-week-old, short day-grown plants that were cold-acclimated for 4 d at 4°C. Error bars represent sd of n = 3 biological replicates. Asterisk indicates significant difference at P < 0.05 (Student’s t test). (D) and (E) Protein levels of V-ATPase and V-PPase increase upon cold exposure in the wild type and UBQ:AVP1. Microsomal membrane proteins of 6-week-old plants were extracted, separated by SDS-PAGE, and subsequently immunoblotted with VHA-C antibody (D) and anti-V-PPase antibody (E). TIP1;1 detection was used as loading control. Protein levels were measured using ImageJ and normalized to TIP1;1. Protein levels at 22°C were set to 100%. Bar charts represent quantification of one representative immunoblot.

Figure 5.

Vacuolar Proton Pump Triple Mutant Is Viable and Has a Neutral Cell Sap pH. (A) fugu5-1 vha-a2 vha-a3 growth is even more inhibited than vha-a2 vha-a3. Wild-type and proton pump mutant plants were grown for 26 d under LD conditions at 22°C. Bar = 1.75 cm. (B) Proton pump triple mutants exhibit defects in flower development. Bright-field micrographs of dissected flowers are shown. Stamen filaments reached the pistil in the wild type and the segregating proton pump triple mutant flower, whereas they are shortened in the fugu5-1 vha-a2 vha-a3 flower. Bar = 1 mm. (C) Silique size of fugu5-1 vha-a2 vha-a3 is strongly reduced. Excised siliques of the wild type, fugu5-1 vha-a2/+ vha-a3, and fugu5-1 vha-a2 vha-a3 are shown. Bar = 3 mm. (D) Cell sap pH of fugu5-1 vha-a2 vha-a3 is nearly neutral. Plants were grown for 3 weeks under LD conditions at 22°C. Error bars show sd of n = 3 biological replicates. (E) to (J) Vacuoles of epidermal cells ([E] to [G]) and mesophyll cells ([H] to [J]) of rosette leaves of 5-week-old wild-type and vacuolar proton pump mutants stained with BCECF (green). Autofluorescence of chloroplasts is shown in magenta. Bars = 25 µm.

Figure 6.

Root Vacuole Morphology in Tonoplast Proton Pump Mutants. (A) to (D) The shape of vacuoles was monitored in the root differentiation zone, elongation zone, and meristematic zone of 6-d-old wild-type (A), vha-a2 vha-a3 (B), fugu5-1 (C), and fugu5-1 vha-a2 vha-a3 (D) seedlings. Roots were stained with BCECF (green) and FM4-64 (red). Bars = 20 µm. (E) to (G) Vacuolar pH measurement in three different root zones. pH values were determined in the root differentiation zone (E), elongation zone (F), and meristematic zone (G) of 6-d-old wild-type and vacuolar proton pump mutant seedlings. Error bars represent sd of n = 3 biological replicates.

Figure 7.

Inhibition of V-ATPase Abolishes Vacuolar Acidification. (A) Inhibition of P-type ATPases has minor effect on vacuolar pH. Vacuolar pH was measured in roots of 6-d-old seedlings after treatment with (+) or without (−) 2 mM vanadate for 18 h. Error bars represent sd of n = 3 biological replicates. (B) ConcA-mediated inhibition of V-ATPase blocks vacuolar acidification. Vacuolar pH was measured in roots of 6-d-old plants. Seedlings of Arabidopsis wild-type and vacuolar proton pump mutants were incubated for 20 h in 1 µM ConcA (+) or DMSO (−) prior to pH measurements. Error bars represent sd of n = 3 biological replicates. (C) Vacuolar morphology after ConcA treatment. Wild-type seedlings were incubated for 20 h in 1 µM ConcA or DMSO. Roots were stained with BCECF (green) and FM4-64. Bars = 20 µm.